In our model of regulation, tropomyosin can exist in two positions on the thin filament, forming either the weak-binding state, or the strong-binding state of regulated actin. In the weak-binding state, which occurs in the absence of Ca2+, troponin-tropomyosin inhibits the binding of S-1 and S-1 ADP to actin, but not S-1.ATP. Therefore, ATPase inhibition seems to be due to regulated actin in the weak-binding state inhibiting Pi release. First, results were obtained consistent with the latter part of our model using S-1 which was cross-linked to actin. Troponin-tropomyosin was found to markedly inhibit the ATPase activity of cross-linked actin S-1 both at low and high ionic strength. Since cross-linked S-1 appears to behave kinetically like S-1 in the presence of infinite actin concentration, the extensive regulation obtained with cross-linked S-1 is not due to troponin-tropomyosin blocking the binding of cross-linked S-1 to actin. Second, we examined the ability of pPDM.S-1, which is a stable analog of S-1.ATP, to turn on or potentiate the ATPase activity of regulated acto S-1. Consistent with our binding data, which shows no apparent cooperatively in the binding of S-1.ATP or pPDM.S-1.ATP to regulated actin, pPDM.S-1 ATP does not significantly turn on the ATPase activity of regulated acto S-1. These results indicate that tropomyosin remains in the inhibitory position when pPDM S-1 ATP binds to regulated actin. Third, we found that in contrast to the results obtained with skeletal muscle, the inhibition of Pi release in the actomyosin ATPase cycle of smooth muscle is not accompanied by inhibition of the binding of myosin.ADP to actin. These results show that although inhibition of Pi release appears to be a general mechanism of muscle regulation, it is not always coupled to inhibition in the binding of myosin ADP to actin.